Chronic inflammation is a fundamental component of atherosclerosis, and is promoted by T-cells specific for antigens within lesions. Work in the applicant's laboratory has established the importance Th1 cell differentiation and B7-family costimulators to pro-atherogenic T cell responses. Although there have been important recent advances in the field of T cell regulation, there has been little application of this knowledge to atherosclerosis. Our preliminary data indicate that there are systemic changes in regulatory T cells (Treg) in the setting of hypercholesterolemia, and there is a potentially important connection between the T cell costimulatory molecule ICOS and Treg activity. The objective of this proposal is to achieve a better understand of the regulatory pathways that can limit pro-atherogenic T cell responses. Three specific Aims are proposed. In Aim 1, we will determine if and how regulatory T cells (Treg) influence pro-atherogenic effector T cells responses and lesion development. We hypothesize that hypercholesterolemia leads to enhancement of both effector CD4+ T cell responses and compensatory systemic Treg responses. Treg will be enumerated in lymphoid tissue and lesions in LDLR KO mice, including FoxP3-GFP/LDLR reporter mice. Functional assays will be performed on Treg isolated from LDLR KO mice, and Treg effects on atherosclerosis and CD4+ T cell responses to atheroantigens will be examined by in vivo adoptive transfer and depletion studies. In Aim 2, we will characterize the mechanisms by which the ICOS on T cells regulates atherosclerotic lesion development and maturation. We hypothesize that ICOS exerts an atheroprotective role by enhancing Treg-mediated suppression of plaque-antigen specific effector T cells and/or by skewing the effector phenotype of plaque-antigen specific effector T cells. We will analyze extent and phenotype of lesions, as well as effector and Treg responses to atheroantigens, in ICOS and ICOS ligand deficient LDLR KO mice. Bone marrow chimeras, and double KO mice will be employed. In Aim 3, we will determine if the PD-L1/PD-L2L-PD-1 T cell regulatory pathway influences the development of atherosclerosis and associated T cell responses. We hypothesize that PD-L1 and/or PD-L2, expressed on bone marrow derived antigen presenting cells or on endothelial cells bind to PD-1 on T cells and function to limit T cell responses to atheroantigens. We will employ LDLR KO mice deficient in PD-L1 and PD-L2 or PD-1, using bone marrow chimeras, and compound KO mice. Atherosclerosis remains a major cause of morbidity and mortality throughout the world. Successful completion of the Aims in this proposal will lead to a better understanding of how immune responses that aggravate atherosclerotic disease may be therapeutically controlled.